Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Schematic representation of experimental procedure. Intestines from wild-type or Rpl24 Bst/+ mice were analysed by histology or processed to make intestinal organoids. ( B ) Staining for H&E, BrdU, RPL24, and P-eEF2 T56 in sections from the small intestines of wild-type and Rpl24 Bst/+ mice. Red brackets in P-eEF2 staining indicate crypts and villi, corresponding to quantification to the right. Bars represent 50 µm. Graphs on the right show scoring for BrdU-positive cells, and H -score calculated for RPL24 and P-eEF2 T-56, plotted ± standard error of the mean (SEM). Significance was determined by one-tailed Mann–Whitney U test. ( C ) Micrographs of small intestinal organoids generated from wild-type or Rpl24 Bst/+ mice. ( D ) Protein synthesis rate quantified by 35 S-methionine incorporation in wild-type or Rpl24 Bst/+ organoids ( n = 3), expressed relative to the wild-type protein synthesis rate (=1). Data are from three biologically independent organoid lines for each genotype represented ± SEM with significance determine by Mann–Whitney U test. ( E ) Representative polysome profiling from wild-type or Rpl24 Bst/+ organoids. Average polysome:subpolysome ratios from three independent organoid lines per genotype are shown above each profile ± SEM. ( F ) Western blotting from protein lysates generated from three biologically independent organoid lines for each genotype. Values for RPL24 expression relative to β-actin and P-eEF2 T56 relative to eEF2 are shown under each lane. The average of the wild-type lanes in both cases has been set to 1. There is a 47% reduction in RPL24 and a 66% increase in P-eEF2 T56 in the Rpl24 Bst/+ organoids. ( G ) Schematic of the potential role of RPL24 in regulating protein synthesis via eEF2. All scale bars are 50 μm. Figure 1—source data 1. Top: data from . Bottom: original western blotting images with areas for each antibody annotated and boxed in red.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Staining, One-tailed Test, MANN-WHITNEY, Generated, Western Blot, Expressing

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Schematic representation of experimental protocols. Villin CreER Apc fl/fl Kras G12D/+ or Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Tissue was taken for histological analysis or processed into intestinal organoids. ( B ) Quantification of BrdU incorporation in small intestinal crypt/villus axes following deletion of Apc and activation of Kras , with ( n = 4) or without ( n = 3) Rpl24 Bst mutation. Data are represented as the mean number of BrdU-positive cells per half crypt/villus from >20 axes per mouse ± standard error of the mean (SEM). Significance was determined by Mann–Whitney U test. ( C ) Representative images of intestines from the same experiment as in ( B ), stained for H&E, BrdU, P-eEF2 T56, and RPL24. The red bar on the H&E images indicates the extent of the proliferative zone. Bars represent 50 µm. ( D ) Apc fl/fl Kras G12D/+ organoids with or without Rpl24 Bst mutation were grown for 4 days and growth relative to day 1 determined by Cell-Titer Blue assay. Data show the mean ± SEM of n = 3 independent organoid lines. Significance was determined by one-tailed Mann–Whitney U test. ( E ) Top: schematic of experimental protocol. Villin CreER Apc fl/+ Kras G12D/+ or Villin CreER Apc fl/+ Kras G12D/+ Rpl24 Bst/+ mice induced with 80 mg/kg tamoxifen then monitored until clinical endpoint. Survival plot for these genotypes for the days post-induction that they reached endpoint. The median survival and n number for each cohort are shown and significance determined by Mantel–Cox test. Censored subjects were removed from the study due to non-intestinal phenotypes. All scale bars are 50 μm.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Injection, BrdU Incorporation Assay, Activation Assay, Mutagenesis, MANN-WHITNEY, Staining, One-tailed Test

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Top: Schematic representation of experimental approach. Villin CreER Apc fl/fl Kras G12D/+ , Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ , or Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ Eef2k D273A/D273A mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Bottom: Staining for P‐4E‐BP1 T37/46, P‐RPS6 S240/4, and P‐eIF2α S51 from the genotypes above. Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ Eef2k D273A/D273A intestines were not stained for P‐eIF2α S51. Right: H‐score quantification for P‐eEF2 T56 (matched to images shown in and ), P‐4E‐BP1 T37/46, P‐RPS6 S240/4, and P‐eIF2α S51 . Small intestines from at least 3three animals from each genotype ( Apc fl/+ Kras G12D/+ , Apc fl/+ Kras G12D/+ Rpl24 Bst/+ , and Apc fl/fl Kras G12D/+ Rpl24 Bst/+ Eef2k D273A/D273A ) were stained and the intensity quantified from the proliferative crypt region. Data are plotted ± standard error of the mean (standard error of the mean, SEM). Significance was determined by one‐way analysis of variance (ANOVA) analysis with Tukey’s multiple comparison. ( B ) Left: Representative micrograph images of each genotype stained for BrdU in the medial colons of Villin CreER Apc fl/fl Kras G12D/+ and Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ mice. Right: Scores for BrdU positivity from 4 mice per genotype, each plotted as the average of at least 20 half crypt/villi. Significance was determined by Mann– Whitney U test.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Injection, Staining, Comparison, MANN-WHITNEY

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Schematic representation of experimental approach. Villin CreER Apc fl/fl Kras G12D/+ or Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Intestines were enriched for crypt epithelium for sucrose density analysis or processed into intestinal organoids. ( B ) Representative sucrose density polysome profiles generated from Apc fl/fl Kras G12D/+ intestinal extracts with or without the Rpl24 Bst mutation. Subpolysomal components (40S, 60S, and 80S) and polysomes are labelled, with the polysomes also split pictorially into light and heavy. ( C ) Quantification of the heavy:light polysome ratio from the experiment in ( B ). Data show the mean of analysis from three mice ± standard error of the mean (standard error of the mean, SEM) with significance determined by one-tailed Mann–Whitney U test. ( D ) Relative protein synthesis rate quantified by 35 S-methionine incorporation in Apc fl/fl Kras G12D/+ three biologically independent organoid lines either wild-type or mutant for Rpl24 Bst . Data are represented ± SEM with significance determine by Mann–Whitney U test. ( E ) Ribosome run-off rate determined in Apc fl/fl Kras G12D/+ small intestinal organoid lines either wild-type or mutant for Rpl24 Bst ( n = 3 per genotype). Data are represented as the mean of three biological replicates ± SEM with significance determine by Mann–Whitney U test. Raw data are available in . ( F ) Schematics of the regulation of protein synthesis and tumour proliferation downstream of RPL24. Smaller RPL24 in bottom scheme represents reduced RPL24 expression. ‘P’ represents phosphorylation of eEF2.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Injection, Generated, Mutagenesis, One-tailed Test, MANN-WHITNEY, Expressing, Phospho-proteomics

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Representative polysome profiles from Apc fl/fl Kras G12D/+ or Apc fl/fl Kras G12D/+ Rpl24 Bst/+ small intestinal organoid cultures, pre-treated with harringtonine for 5 min/300 s (H300) or untreated (H0). These traces were analysed for the run-off rates shown in . ( B ) Representative polysome profiles from Apc fl/+ Kras G12D/+ or Apc fl/+ Kras G12D/+ Rpl24 Bst/+ colonic adenoma cultures (left) and quantification of the polysome to subpolysome ratio from these (right). Two biologically independent lines were analysed per genotype and plotted ± standard error of the mean (SEM). Scheme above denotes the generation of adenoma cultures from distinct colonic tumours in aged Villin CreER Apc fl/+ Kras G12D/+ and Villin CreER Apc fl/+ Kras G12D/+ Rpl24 Bst/+ mice. ( C ) Relative protein synthesis rates quantified by 35 S-methionine incorporation in the colonic adenoma cultures described in ( B ) with n = 3. The average protein synthesis rates were plotted relative to Apc fl/+ Kras G12D/+ controls (=1) for three organoid lines per genotype ± SEM. Significance was determined by Mann–Whitney U test. ( D ) 60S to 40S ratio from sucrose density gradients from lysates generated from the indicated genotypes. Data show the mean ± SEM. Representative traces are shown in and . ( E ) Western blotting from protein lysates generated from three biologically independent organoid lines for each genotype. Mean values for RPL24, RPL10, RPL22, and RPS6 expression relative to β-actin and P-eEF2 T56 relative to eEF2 are shown next to the relevant blot ± SEM. The average of the Villin CreER Apc fl/+ Kras G12D/+ lanes in all cases has been set to 1.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: MANN-WHITNEY, Generated, Western Blot, Expressing

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Schematic representation of experimental approach. Villin CreER Apc fl/fl or Villin CreER Apc fl/fl Rpl24 Bst/+ mice were induced by two intraperitoneal injection of tamoxifen at 80 mg/kg on days 0 and 1 then intestinal tissue analysed on day 4. Intestines were analysed histologically or intestinal organoids generated. ( B ) Top: representative micrographs showing proliferation as BrdU positivity and extent of proliferation as a red bar in H&E image. RPL24 and P-eEF2 T56, staining is also shown for each genotype. Bars represent 50 µm. Below: BrdU scoring from Apc fl/fl or Apc fl/fl Rpl24 Bst/+ mouse intestines and H -scores for RPL24 and P-eEF2 T56 protein levels. For BrdU scoring BrdU was administered 2 hr before sampling and at least 20 half crypt/villus axes were scored per animal and the mean plotted ± standard error of the mean (standard error of the mean, SEM). ( C ) Apc Min/+ tumour model survival curve, for mice with and without Rpl24 Bst mutation. Lack of a significant difference was determined by Mantel–Cox test. ( D ) Relative growth of Apc fl/fl and Apc fl/fl Rpl24 Bst/+ small intestinal organoids over 3 days, measure by Cell-Titer Blue assay. The average change in proliferation is plotted from three independent biological replicates per genotype. ( E ) Relative protein synthesis rates quantified from 35 S-methionine incorporation into Apc fl/fl , Apc fl/fl treated with 250 nM rapamycin for 24 hr and Apc fl/fl Rpl24 Bst/+ small intestinal organoids. Significant changes were calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype with the mean protein synthesis rate for each genotype plotted ± SEM. ( F ) Relative expression of ribosomal protein mRNAs in Villin CreER Apc fl/fl and Villin CreER Apc fl/fl Kras G12D/+ whole intestine samples, where wild-type tissue has been normalised to 1. The fold increase in expression from Apc fl/fl to Apc fl/fl Kras G12D/+ samples for Rpl24 and the average of all other RP mRNAs are shown. Statistical analysis was by one sample t -test of the other RP mRNA fold changes using the fold change for Rpl24 mRNA as the hypothetical mean. All scale bars are 50 μm. Figure 4—source data 1. Data relate to and . Raw values tab shows the tabulated RNA sequencing reads for ribosomal proteins from triplicate wild-type (WT), Villin CreER Apc fl/fl (Apc) and Villin CreER Apc fl/fl Kras G12D/+ (Apc Kras) from whole intestine samples. Averages for the replicates are shown and fold change compared to WT expression. Average values tab shows all fold changes and the average fold change across all ribosomal proteins except Rpl24 , and the fold change for Rpl24 for comparison.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Injection, Generated, Staining, Sampling, Mutagenesis, Comparison, Expressing, RNA Sequencing

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Schematic representation of experimental approach. Villin CreER Apc fl/fl Kras G12D/+ , Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ , or Villin CreER Apc fl/fl Kras G12D/+ Rpl24 Bst/+ Eef2k D273A/D273A mice were induced by intraperitoneal injection of tamoxifen at 80 mg/kg then intestinal tissue analysed 3 days later. Intestines were analysed histologically or processed into intestinal organoids. ( B ) BrdU incorporation quantified from within small intestinal crypt/villus axes following deletion of Apc and activation of Kras , either wild-type of mutant for Rpl24 , or mutant for Rpl24 and Eef2k . Data are represented as the mean of at least 20 crypt/villi per mouse ± standard error of the mean (SEM) with significance determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype. ( C ) Representative images of H&E, BrdU, and P-eEF2 T56 staining of intestines from the same experiment as ( B ). Red bar on H&E indicates extent of proliferative zone. Bars represent 50 µm. ( D ) Organoids deficient for Apc and with activated Kras with or without Rpl24 Bst mutation, or mutant for both Rpl24 and Eef2k were grown for 3 days and growth relative to day 1 determined by Cell-Titer Blue assay. Data show the mean ± SEM of n = 3 biologically independent organoid lines. Significance was determined by one-tailed Mann–Whitney U test. ( E ) Survival plot for Apc Kras ageing mice with or without the Rpl24 Bst mutation, Eef2K mutation and with both Rpl24 and Eef2k mutations. Median survival and n numbers for each cohort are shown and significance determined by Mantel–Cox test. Censored subjects were removed from the study due to non-intestinal phenotypes. All scale bars are 50 μm.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Injection, BrdU Incorporation Assay, Activation Assay, Mutagenesis, Comparison, Staining, One-tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) 35 S-methionine incorporation to determine relative protein synthesis by in Apc fl/fl Kras G12D/+ small intestinal organoids wild-type or mutant for Rpl24 or with both Rpl24 andConsistent with this, the Eef2k mutations. Data are represented ± standard error of the mean (standard error of the mean, SEM) with significance determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype, each representing an independent organoid line. ( B ) Ribosome run-off rate determined in Apc fl/fl Kras G12D/+ small intestinal organoids mutant or wild-type for Rpl24 or with both Rpl24 and Eef2k mutations. Data are the mean of three biologically independent organoid lines represented ± SEM with significance determined by Mann–Whitney U test. Raw data are available in . The run-off rate for Apc fl/fl Kras G12D/+ control organoids is reproduced from . ( C ) Schematic representation of findings in Apc -deficient Kras- mutant mouse and organoid models. Top: RPL24 expression maintains translation and proliferation by suppressing the phosphorylation of eEF2 by limiting eEF2K activity. Middle: reduced expression of RPL24 activates eEF2K, increasing P-eEF2, reducing translation elongation and suppressing tumorigenesis and proliferation. Bottom: inactivation of eEF2K reverts the phenotype in Rpl24 Bst cells, due to the inability to phosphorylate and suppress eEF2. Elevated elongation rates correlate with increased proliferation following inactivation of eEF2K.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Mutagenesis, Comparison, MANN-WHITNEY, Control, Expressing, Phospho-proteomics, Activity Assay

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Schematic of the findings presented here from pre-clinical mouse models. KRAS activation requires RPL24 expression to maintain low eEF2 phosphorylation. This occurs via a double negative regulation of eEF2K, whereby RPL24 suppresses eEF2K, which suppresses eEF2. eEF2 activity correlates with protein synthesis and proliferation rates. Dashed lines indicate indirect or undefined regulatory pathways. ( B ) RNA expression levels of RPL24 , EEF2K , and EEF2 between normal colon and colon adenocarcinoma samples using data extracted from The Cancer Genome Atlas by TNMplot. Relative expression changes are annotated, as well as p values for each transcript.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Activity Assay, RNA Expression

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet: ( A ) Protein levels of RPL24, eEF2K, and eEF2 in normal colon and colon adenocarcinoma collated from the Clinical Proteomic Tumor Analysis Consortium by UALCAN. Numbers of samples and p values are shown for each protein. ( B ) RNA expression levels of RPL24 , EEF2K , and EEF2 between normal rectum and rectum adenocarcinoma samples using data extracted from The Cancer Genome Atlas by TNMplot. Relative expression changes are annotated, as well as p values for each transcript.

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: RNA Expression, Expressing

Journal: eLife

Article Title: Rpl24 Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

doi: 10.7554/eLife.69729

Figure Lengend Snippet:

Article Snippet: IHC staining was carried out as previously , using the following antibodies: BrdU (BD Biosciences #347580), P-eEF2 T56 (Cell Signaling Technology [CST] #2331), P-4E-BP1 T37/46 (CST #2855), P-RPS6 S240/4 (CST #5364), P-eIF2α S51 (CST #3398), P-ERK T202/Y204 (CST #9101), RPL24 (Sigma-Aldrich HPA051653), and Lysozyme (Dako A0099).

Techniques: Sequencing, RNAscope, Recombinant, Plasmid Preparation, Labeling, Software